1. Technical Field
The present disclosure relates to a method and a device for detecting a gene (DNA).
2. Description of the Related Art
There is a method in which a desired DNA/RNA fragment of a gene is amplified to an amount necessary for detection by a technique called PCR (Polymerase Chain Reaction) to detect the desired DNA/RNA fragment. Further, a quantitative analysis technique called qPCR (quantitative PCR) is often used as an advanced type of PCR. Such a quantitative gene analysis technique is introduced in a compact reactor or the like provided in a microchannel chip.
Patent Literature 1 discloses a basic method for implementing qPCR for quantitative gene analysis. According to Patent Literature 1, a sample containing a single-stranded DNA is brought into contact with an oligonucleotide (short DNA/RNA sequence) having a sequence complementary to a first region of a target DNA sequence and a labeled oligonucleotide containing a sequence complementary to a second region of the same target DNA sequence. A double-stranded complex mixture is prepared under conditions where hybridization occurs, and the annealed labeled oligonucleotide is cleaved by 5″→3′ nuclease activity to liberate a labeled fragment. A method for detecting such a liberated labeled fragment is disclosed. When the labeled oligonucleotide is prepared by labeling with a fluorescent dye and a quencher, fluorescence is not emitted until a labeled fragment is liberated, and fluorescence intensity is increased by repeating the above process. The fluorescence intensity is detected with a photodetector to analyze how much the target DNA sequence of interest is contained. When DNA/RNA sequences of two or more regions need to be detected per sample, labeled oligonucleotides complementary to the different DNA/RNA sequences are prepared by labeling with different fluorescent dyes that emit fluorescence of different wavelengths. Such a difference in fluorescence wavelength makes it possible to separately analyze the DNA/RNA sequences with a photodetector.
Patent Literature 2 discloses one example of a method for implementing a quantitative gene analysis technique. Particularly, Patent Literature 2 discloses a technique for improving a high-throughput assay using an emulsification technique. The emulsification technique is used to generate droplets that function as independent reaction chambers for biochemical reactions, and individual sub-components (e.g., cells, nucleic acids, and proteins) are processed and assayed using the droplets.
Aqueous droplets containing DNA/RNA or the like are suspended in oil to prepare an emulsion in which water is dispersed in oil. This emulsion is stabilized with a surfactant so that coalescence of droplets during heating, cooling, or transport can be reduced or prevented. This makes it possible to perform thermal cycling. For this reason, emulsions are used to perform single-copy amplification of nucleic acid target molecules in droplets using PCR. Among these droplets, those positive for a target can be analyzed based on Poisson statistics to estimate a concentration of the target in a sample. Droplet-based assays use one or more fluorophores as labels in droplets to determine the occurrence of a reaction such as amplification. Droplets are generated and reacted, and then light emitted from each of the droplets is measured, which makes it possible to determine whether or not a target is present in the droplet. When different distinguishable fluorophores are used for different targets, the presence or absence of two or more different targets can be measured in each droplet. In such a case where two or more different targets need to be distinguished, two or more fluorophores, that is, dye materials that emit fluorescence of different wavelengths are often used to distinguish the targets based on their fluorescence wavelengths. Patent Literature 2 discloses a method for distinctively detecting two fluorophores used. The method is achieved by providing different detection systems (including a light source and a detector) for first and second dyes so that the detection system for the first dye and the detection system for the second dye alternately detect droplets when the droplets pass through an examination region of a channel.